Unfolding titin immunoglobulin domains

The giant muscle protein titin, also known as connectin, is a roughly
30,000 amino acid long filament which plays a number of important roles
in muscle contraction and elasticity (Labeit et al., 1997; Maruyama,
1997; Wang et al., 1993). Titin has been connected with the diseases
myasthenia gravis (Lubke et al., 1998) and hypertrophic cardiomyopathy
(Rottbauer et al., 1997). The I- band region of titin, largely composed
of immunoglobulin-like (Ig) domains, is believed to be responsible
for the molecule's extensibility and passive elasticity. Recently
accomplished AFM (Rief et al., 1997) and optical tweezers (Kellermayer
et al., 1997; Tskhovrebova et al., 1997) experiments directly measured
the force-extension profile of single titin molecules. In the AFM
experiment, cloned sections of titin composed of adjacent I-band Ig
domains were stretched at constant speed. The force-extension profile
showed a sawtooth-shaped pattern with about 250 to 280 A spacing between
the force peaks, with every force peak corresponding to a single Ig
domain unfolding. The Ig domains were thus observed to unfold one
by one, as opposed to concurrently, under the influence of applied
external force Figure 1). To examine in atomic detail the dynamics and
structure-function relationships of this behavior, SMD simulations of
force-induced titin Ig domain unfolding were performed [1,3,7].

Figure 1

Figure 2

The SMD simulations were performed with an NMR structure of the Ig
domain I27 of the cardiac titin I-band (Improta et al., 1996). I27
consists of two beta-sheets packed against each other, with each sheet
containing four strands, as shown in Fig. 1b. The domain was solvated
and equilibrated, then an SMD simulation was carried out by fixing one
terminus of the domain and applying a force to the other in the direction
from the fixed terminus to the other one. Simulations were performed
following the scheme of F = K (vt - x) with v = 0.5 A/ps and 0.1 A/ps and
K = 10 kB T /A2 at 300 K. The recorded force-extension profile from the
SMD trajectory (see Figure 2) showed a single force peak at the initial
stage of the Ig domain extension. This feature agrees well with the
sawtooth-shaped force profile exhibited in the AFM experiment.

Examination of the details of the simulation trajectory provides
an explanation of how the early force maximum was produced (see Figure
3). Initially (0-10 A extension), the two beta-sheets slid away from each
other, each maintaining a stable structure as well as its intra-sheet
backbone hydrogen bonds. As the extension of the domain reached 14 A,
the structure within each sheet began to break: in one sheet, strands
A' and G slid past each other, while in the other sheet, strands A and
B slid past each other. The A'-G and A-B hydrogen bonds broke nearly
simultaneously, producing the large initial force peak seen in Figure
2. These events marked the beginning of the Ig domain unraveling, after
which the domain gradually unfolded and strands unraveled one at a
time, accompanied by a large reduction in the recorded force. After
an extension of 260 A, the domains were completely unfolded; further
extension stretched the already extended polypeptide chain and caused the
force to dramatically increase.

Figure 3

Figure 4

Constant force stretching simulations, applying 500 - 1000 pN of
force, were also performed [3,4,6,7]. The resulting domain extensions are
halted at an initial extension of 10 A until the set of all six hydrogen
bonds connecting terminal beta-strands break simultaneously. This
behavior is accounted for by a barrier separating folded and unfolded
states, the shape of which is consistent with AFM and chemical
denaturation data. Detailed analysis of protein water interaction shows
that the breaking of hydrogen bonds between strands A' and G needs to be
assisted by fluctuations of water molecules. In nanosecond simulations,
water molecules are found to repeatedly interact with the protein
backbone atoms, weakening individual inter-strand H-bonds until all six
A'G hydrogen bonds break simultaneously under the influence of external
stretching forces. Only when those bonds are broken can the generic
unfolding take place, which involves hydrophobic interactions of the
protein core and exerts weaker resistance against stretching [7].

The simulation suggests how Ig domains achieve their chief design
requirement of bursting one by one when subjected to external forces. At
small extensions, the hydrogen bonds between strands A and B and between
strands A' and G prevent significant extension of a domain, i.e., the
domain maintains its beta-sandwich structure. After these bonds break,
resistance to unfolding becomes much smaller, and the domain unfolds
rapidly. Only when a domain is fully extended does the force increase
enough to begin the unfolding process in another domain.

Combining atomic force microscopy data with SMD simulation resulted
in the discovery of a mechanical unfolding intermediate in titin Ig
domain I27 [6]. During AFM extension of a multimer of Ig domains,
before the first domain unfolding event took place, every domain was
observed to extend by 6 Angstoms. SMD simulations showed that the rupture
of a pair of hydrogen bonds near the amino terminus of an Ig domain
(while the other inter-strand hydrogen bonds remain intact) allows
this extension. Disruption of these hydrogen bonds by site-directed
mutagenesis eliminates the unfolding intermediate.

Figure 5

SMD simulation of forced unfolding of fibronectin type III (FnIII)
domain (similar to the non-Ig repeated titin domains) and of other types
of protein domains have also been performed [2,3]. The behavior of the
proteins under external forces can be classified into 2 classes. Class I
domains exhibit high resistance to forced unfolding; their fold topology
are such that inter-strand hydrogen bonds must break in clusters in
order to allow extension of the domain. This class includes titin Ig and
FnIII domains. Class II domains, for example all-helix domains, have
topologies that can be extended while breaking inter-strand hydrogen
bonds singly, they do not exhibit dominant force peaks when stretched in
SMD simulations.